Class of near infrared optical probes for biological applications

ABSTRACT

2,5-Bis(benzoxazol-2′-yl)benzene-1,4-diol derivatives (Zinhbo derivatives) are used to detect zinc ions and have particular application in vivo and in vitro. Zinhbo derivatives upon excitation give a florescence response emission that can be used to determine the presence of zinc cation in solution. Zinhbo derivatives complexed with zinc cations upon excitation can produce a florescence response emission in the visible and near infrared range. Zinhbo derivatives complexed with zinc cations exhibit a large stoke shift between the excitation and emission wavelengths.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from the pending U.S. patentapplication Ser. No. 13/622,109, filed on Sep. 18, 2012, which claimspriority to U.S. Provisional Patent Application No. 61/493,572, thecontents of both being incorporated herein by reference.

FIELD OF THE INVENTION

The present invention generally relates to compounds for the detectionof zinc ions, the synthesis of compounds for the detection of zinc ions,compositions for the detection zinc ions, and methods for detecting zincions.

BACKGROUND OF THE INVENTION

Organic fluorescent probes are useful labeling for biomolecules. For invivo applications, molecular imaging reagent is required to bebiocompatable and to emit optical signals in the near infrared (NIR)region (700-900 nm), as NIR light can penetrate more deeply intobiological tissues. On the practical side, optical imaging is dependenton the availability of the luminescent NIR reagents that exhibit highquantum yield, chemical and optical stability, and suitablepharmacological properties including aqueous solubility, specificbinding, and low toxicity. At present time, most of the NIR probes arebased on cyanine dyes, whose emission maxima are in the region of650-900 nm. A notable drawback for the parent cyanine dyes is theirsmall Stokes shift (typically about 20-50 nm), which hampers their broadapplication.

Among the new emerging design principles applied in fluorescent sensing,excited-stated intramolecular proton transfer (ESIPT) has recentlyreceived considerable attention due to its unique photophysicalproperties. Different from other organic chromophores, ESIPT moleculesexhibit dual emissions from both the excited enol and keto tautomers,which are well separated from each other. In addition, emission of ESIPTdyes generally have large Stokes shift (ca. 150-200 nm), making them theideal candidates for fluorescent sensors. Some ESIPT-based molecules,including 2-(2′-hydroxyphenyl)benzoxazole (HBO) and2-(2′-hydroxyphenyl)benzimidazole (HBI), have been reported for cationsand anion sensing. Most studies utilize ESIPT turn-off mechanisms sincethe interaction with a cation (or anion) removes the phenolic proton,thereby inhibiting ESIPT and resulting in blue-shifted fluorescence.Removal of the phenolic proton during metal chelation, however,permanently turns-off ESIPT. Thus far, only a few examples are known toutilize ESIPT turn-on mechanism in the chemosensor design, whichinvolves the deprotection of the protected hydroxyl group. Among theknown examples, nearly all ESIPT-based probes give emission in thevisible region (400-650 nm).

As the second most abundant transition-metal ion in the human body, theZn²⁺ ion is a component of enzymes and proteins, and plays an importantrole in various biological processes. In order to discover the vitalroles of Zn²⁺ in biological processes, there is growing demand forsensing Zn²⁺ in living systems. Although many fluorescent chemosensorsfor Zn²⁺ cation have been studied, few near-infrared (NIR) fluorescentzinc probes are available to give emission in the desired 700-900 nmrange. An ideal Zn²⁺ probe requires not only NIR emission (to minimizeautofluorescence) but also large Stokes shift (for improved signaldetection). It is thus desirable to incorporate the ESIPT process intothe sensing scheme. Achieving the ESIPT emission signals in the NIRregion, however, remains an attractive and challenging task.

SUMMARY OF THE INVENTION

One embodiment of this invention provides a composition for detection ofzinc ions defined by the formula:

where R¹ is a hydrogen atom or an electron withdrawing group; R², R³,and R⁴ are each individually selected from the group consisting of ahydrogen atom, an electron withdrawing group, and an electron donatinggroup; and R⁵ is a chelator group capable of forming a complex with azinc cation.

Yet another embodiment provides a method for detecting the presence ofzinc ions in solution, the method comprising the steps of contacting asolution with a Zinhbo derivative defined by the formula:

where R¹ is a hydrogen atom or an electron withdrawing group; R², R³,and R⁴ are each individually selected from the group consisting of ahydrogen atom, an electron withdrawing group, and an electron donatinggroup; and R⁵ is a chelator group capable of forming a complex with azinc cation, exciting the solution with an excitation wavelength, andmeasuring a florescence response emission at least one of the visible ornear infrared ranges.

Yet another embodiment provides a method for detecting the presence ofzinc ions, the method comprising the steps of contacting living cells ora living organism with a Zinhbo derivative, exciting the solution withan excitation wavelength, and measuring a florescence response emissionat least one of the visible or near infrared ranges

This invention also provides a method as in paragraph [0008] wherein theZinhbo derivative defined by the formula:

where R¹, R², R³, and R⁴ are each individually selected from the groupconsisting of a hydrogen atom, an electron withdrawing group, and anelectron donating group; and R⁵ is a chelator group capable of forming acomplex with a zinc cation.

This invention also provides a method as in paragraph [0010] wherein theZinhbo derivative includes an R², R³, and R⁴ that are hydrogen atoms andan R¹ that is and electron donating group.

This invention also provides a method as in paragraph [0011] wherein theelectron donating group is an alkyl group.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 provides a general schematic of a method for testing one or moretest solutions for the presence of zinc cation in accordance with thisinvention.

FIG. 2 provides a general reaction scheme for the preparation of aZinhbo derivative according to one or more embodiments of thisinvention.

FIG. 3 provides the electron flow for the excited state intramolecularproton transfer (ESIPT) for two embodiments of Zinhbo molecules.

FIG. 4 provides the molecular structure of an HBO unit.

FIG. 5 provides the electron flow for the excited state intramolecularproton transfer (ESIPT) for various HBO and Zinhbo molecules.

FIG. 6 provides the synthesis scheme of an embodiment of the invention,Zinhbo-5 sensor, under the following reagents and conditions: a) (i)MeOH, 5 A Molecular Sieves, Reflux, 81.8%; (ii) DDQ, CH₂Cl₂, 86.9%; b)PCC, CH₂Cl₂; c) (i) Toluene, Reflux, (ii) iodobenzene diacetate (IBD),CH₂Cl₂, 64.6%; d) NBS, AIBN, CCl₄; e) DPA, Na₂CO₃, THF, 12.1%; f) BBr₃,CH₂Cl₂, 17.0%.

FIG. 7 provides a graph of the UV-vis spectra of an embodiment of theinvention, Zinhbo-5 (1.0×10⁻⁵ M), in EtOH upon addition of zinc ions.

FIG. 8 provides a graph of a 300 MHz ¹H NMR titration of Zinhbo-5 inCD₃OD/D₂O (1:1, V/V) with Zn(ClO₄)₂. The t-butyl signal (as a singlet at1.3 ppm) is not shown. The starred signals at 4.80 and 3.29 ppm areattributed to the D₂O and CD₃OD residues, respectively.

FIG. 9 provides a proposed metal complex formation from Zinhbo-5 andZn²⁺ cation. The inset of FIG. 9 shows the geometry-optimized structurefor “(Zinhbo-5)₂-Zn”9 (without t-butyl substituent, using HyperChemMM+). Two pyridyl groups on the back side are shown in the orange colorfor clarity. The thick arrows point to the methylene protons that becomediastereotopic.

FIG. 10 provides a ORTEP plot of the crystal structure of Zinhbo-5-Zn²⁺(10) with the thermal ellipsoids drawn at 50% probability.

FIG. 11 provides a graph of a 500 MHz ¹H NMR spectra of crystal 5 (firsttrace at the top) and “Zinhbo-5+Zn²⁺ (1:1 molar ratio)” in CD₃OD. Thedescriptions in the parentheses indicates the time after mixing Zinhbo-5with Zn²⁺ cation. All spectra use CD₃OD solvent signal at ˜4.8 ppm (notshown) as reference.

FIG. 12 provides a graph of the change in the fluorescence spectra forZinhbo-5 (1.0×10⁻⁵ M) in EtOH with the addition of zinc ions.

FIG. 13 provides a graph of the change in the emission spectra forZinhbo-5 (1.0×10⁻⁵ M) in HEPES buffer solution containing 50% EtOH withthe addition of zinc ions with excitation at 480 nm.

FIG. 14 provides the fluorescence spectra of Zinhbo-5 and Zinhbo-1(1.0×10⁻⁵ M) upon addition of zinc cation in THF (excitation at 480 nm).(Zinhbo-5)₂-Zn²⁺ (2:1 ligand-to-metal ratio) is the predominant formwhen Zn²⁺ is added to Zinhbo-5 in solution.

FIG. 15 provides a schematic representation of the ESIPT process ofZinhbo-5-Zn complex, involving the enol emission at 546 nm and ketoemission at 763 nm. The thick arrows indicate the hydrogen bonds in theexcited states.

FIG. 16 provides a graph of the excitation spectra for Zinhbo-5(1.0×10⁻⁵ M) in HEPES buffer solution containing 50% EtOH with andwithout 1 equivalent of zinc ions.

FIG. 17 provides the fluorescence spectra for Zinhbo-5 (1.0×10⁻⁵ M) indifferent solvents with 1 equivalent of zinc ions.

FIG. 18 provides a graph of the fluorescence intensity of Zinhbo-5 (10μM) at 535 nm as a function of concentration of Zn²⁺ (0-4 μM) inH₂O/EtOH (5/5) solution (containing 50 M HEPES, 0.1M KNO₃, pH=7.2). Theinset of FIG. 18 provides the emission spectra of Zinhbo-5 in thepresence of different concentrations of Zn²⁺ ion. The linearly dependentcoefficient is: R²=0.99978.

FIG. 19 provides a graph of the fluorescence intensity of Zinhbo-5 (10μM) at 710 nm as a function of concentration of Zn²⁺ (0-4 μM) inH₂O/EtOH (5/5) solution (containing 50 M HEPES, 0.1M KNO₃, pH=7.2). Theinset of FIG. 19 provides the emission spectra of Zinhbo-5 in thepresence of different concentrations of Zn²⁺ ion. The linearly dependentcoefficient is: R²=0.9982.

FIG. 20 provides the fluorescence images of Hela cells incubated with 10μM Zinhbo-5 for 30 min (b) and then further incubated with 20 μM Zn²⁺for 30 min (d) and then subsequent addition of 2 mM TPEN (f). Thebright-field transmission image of cells corresponding to their imagesare: (a, c, e; a→b, c→d, e→f).

FIG. 21 provides Zinhbo-5 labeling of developing zebrafish at 17 and 50hours post fertilization (hpf).

FIG. 22 provides that Zinhbo-5 labeling is greatly reduced in zebrafishlarvae (82 hpf) exposed to TPEN, which is a zinc chelator.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

In one or more embodiments, the invention relates to2,5-bis(benzoxazol-2′-yl)benzene-1,4-diol derivatives for the detectionof zinc ions. In other embodiments, the invention relates tocompositions for the detections of zinc ions. In still otherembodiments, the invention relates to methods for the detection of zincions using 2,5-bis(benzoxazol-2′-yl)benzene-1,4-diol derivatives. Thesecompositions and methods will have particular application in vivo and invitro.

The applicants have found that 2,5-bis(benzoxazol-2′-yl)benzene-1,4-diolderivatives can give turn-on excited state intramolecular protontransfer (ESIPT) fluorescence upon addition of zinc cations. A uniquefeature of 2,5-bis(benzoxazol-2′-yl)benzene-1,4-diol derivatives is thatthe sensor molecule contains two 2-(2′-hydroxyphenyl)benzoxazole units.In its zinc complex only one 2-(2′-hydroxyphenyl)benzoxazole unit bindsto a zinc cation, while the other 2-(2′-hydroxyphenyl)benzoxazole unitis retained for ESIPT. Zinc binding not only turns on the fluorescencebut also enables the ESIPT emission with a large Stokes shift. In one ormore embodiments, the 2,5-bis(benzoxazol-2′-yl)benzene-1,4-diolderivative binds a zinc cation that is a Zn²⁺ cation. For furtherdiscussion on florescence of 2,5-bis(benzoxazol-2′-yl)benzene-1,4-diolderivatives see Y. Xu, Y. Pang, Chem. Commun., 46 4070-4072 (Apr. 15,2010) and Y. Xu, Y. Pang, Dalton Transactions, 40 1503-1509 (Feb. 1,2011) which are incorporated in thier entirety by referance.

In one or more embodiments, that2,5-bis(benzoxazol-2′-yl)benzene-1,4-diol derivatives are compoundswhich include a dihydroxyphenyl group bound to two benzoxazole groups.In one or more embodiments, a benzoxazole group of the2,5-bis(benzoxazol-2′-yl)benzene-1,4-diol derivative may include one ormore groups that form a complex with a zinc ion. In one or moreembodiments, a benzoxazole group of the2,5-bis(benzoxazol-2′-yebenzene-1,4-diol derivative may include one ormore substituents that are electron donating groups. In these or otherembodiments, a benzoxazole group of the2,5-bis(benzoxazol-2′-yl)benzene-1,4-diol derivative may include one ormore substituents that are electron withdrawing groups. A2,5-bis(benzoxazol-2′-yl)benzene-1,4-diol derivative may also beconsidered a compound which includes two 2-(2′-hydroxyphenyl)benzoxazoleunits that share a dihydroxyphenyl group. A2-(2′-hydroxyphenyl)benzoxazole unit may also be referred to as a HBOunit (FIG. 4). 2,5-Bis(benzoxazol-2′-yl)benzene-1,4-diol derivatives maybe referred to as Zinhbo derivatives.

In one or more embodiments, the Zinhbo derivative may be defined by thefollowing formula 1:

where R¹, R², R³, and R⁴ are each individually selected from the groupconsisting of a hydrogen atom, an electron withdrawing group, and anelectron donating group; and R⁵ is a chelator group capable of forming acomplex with a zinc cation.

In one or more embodiments, the R¹ of formula 1 is not an electrondonating group. In these and other embodiments, R¹ of formula 1 is notan alkyl group. In these or other embodiments, R¹ of formula 1 is anelectron withdrawing group or a hydrogen atom. Though not to be bound byany particular theory, it is believed that, when R¹ of formula 1 is anelectron donating group the electron flow required for the excited-stateintramolecular proton transfer is partially perturbed by the electrondonating effect (FIG. 3).

Electron donating groups, also referred to as activating groups, aregroups that add electron density to the benzene ring. Electron donatinggroups are typically classified by their strength into groups consistingof strong electron donating groups, moderate electron donating groups,and weak electron donating groups.

Examples of strong electron donating groups include, but are not limitedto, an alcohol group (—OH), an oxyl group (—O⁻), an amino group (—NH₂),alkylamino groups (—NHR), and dialkylamino groups (—NR₂). Examples ofmoderate electron donating groups include, but are not limited to,alkoxy groups (—OR) and amide groups (—NHCOR). Examples of weak electrondonating groups include, but are not limited to, alkyl groups (—R). Forthe purpose of defining donating groups R can be defined as an alkylgroup. In one or more embodiments alkyl groups include linear orbranched hydrocarbons with a carbon chain length of 1 to 6 carbons.Specific examples of alkyl groups include, but are not limited to,methyl, ethyl, propyl, isopropyl, isobutyl, tert-butyl, n-butyl,sec-butyl, isopentyl, tertpentyl, n-pentyl, sec-pentyl, terthexyl,n-hexyl, isohexyl, and sec-hexyl.

Specific examples of alkylamino groups suitable for use as an electrondonating group include, but are not limited to, methylamino, ethylamino,propylamino, isopropylamino, isobutylamino, tert-butylamino,n-butylamino, sec-butylamino, isopentylamino, tertpentylamino,n-pentylamino, sec-pentylamino, terthexylamino, n-hexylamino,isohexylamino, and sec-hexylamino. Specific examples of dialkylaminogroups suitable for use as an electron donating group include, but arenot limited to, dimethylamino, diethylamino, dipropylamino,diisopropylamino, diisobutylamino, di-tert-butylamino, di-n-butylamino,di-sec-butylamino, diisopentylamino, tertpentylamino, di-n-pentylamino,di-sec-pentylamino, di-tert-hexylamino, n-hexylamino, diisohexylamino,di-sec-hexylamino, methylethylamino, methylpropylamino,methylisopropylamino, methylisobutylamino, tert-butylmethylamino,n-butylmethylamino, ethylpropylamino, ethylisopropylamino,ethylisobutylamino, tert-butylethylamino, and n-butylethylamino.

In one or more embodiments, alkyl groups suitable for use as an electrondonating group include linear or branched hydrocarbons with a carbonchain length of 1 to 6 carbons. Specific examples of alkyl groupssuitable for use as an electron donating group include, but are notlimited to, methyl, ethyl, propyl, isopropyl, isobutyl, tert-butyl,n-butyl, sec-butyl, isopentyl, tertpentyl, n-pentyl, sec-pentyl,terthexyl, n-hexyl, isohexyl, and sec-hexyl.

Specific examples of alkoxy groups suitable for use as an electrondonating group include, but are not limited to, methoxy, ethoxy,propoxy, isopropoxy, isobutoxy, tert-butoxy, n-butoxy, sec-butoxy,isopentoxy, tertpentoxy, n-pentoxy, sec-pentoxy, terthexoxy, n-hexoxy,isohexoxy, and sec-hexoxy.

Specific examples of amide groups suitable for use as an electrondonating group include, but are not limited to, acetamide, propanamide,butyramide, isobutyramide, pentanamide, isopentanamide, andtertpentanamide.

Electron withdrawing groups, also referred to as deactivating groups,are groups that remove electron density from the benzene ring. Electronwithdrawing groups are typically classified by their strength intogroups consisting of strong electron withdrawing groups, moderateelectron withdrawing groups, and weak electron withdrawing groups.

Examples of strong electron withdrawing groups include, but are notlimited to, a nitro group (—NO₂), quaternary amine groups (—NR₃), andtrihalomethane groups (—CX₃). Examples of moderate electron withdrawinggroups include, but are not limited to, a cyano group (—CN), a sulfonategroup (—SO₃H), a carboxylic acid group (—COOH), ester groups (—COOR), analdehyde group (—CHO), and ketone groups (—COR). Examples of weakelectron withdrawing groups include, but are not limited to, halideatoms (—X). For the purpose of defining electron withdrawing groups Rcan be defined as an alkyl group described above.

Specific examples of ester groups suitable for use as an electronwithdrawing group include, but are not limited to, methanoate,ethanoate, propanoate, butanoate, pentanoate, and hexanoate.

Specific examples of ketone groups suitable for use as an electronwithdrawing group include, but are not limited to, ethanoyl, propanoyl,butanoyl, pentanoyl, and hexanoyl.

Specific examples of halide atoms suitable for use as an electronwithdrawing group include, but are not limited to, a fluoride atom, achloride atom, a bromide atom, an iodide atom, and an astatide atom.

In one ore more embodiments, the chelator group capable of forming acomplex with a zinc cation include linear or cyclic polyamine chelatinggroups. Specific examples of groups that form complexes with zinc ionsinclude, but are not limited to, linear polyamine chelators such asdi-2-picolylamine, and cyclic polyamine chelators such as1,4,7,10-tetrazazcyclododecane.

In one or more embodiments, 3 of R¹, R², R³, and R⁴ of formula 1 arehydrogen atoms. In particular embodiments, where R¹, R³, and R⁴ offormula 1 are hydrogen atoms, the Zinhbo derivative may be defined bythe following formula 2:

where R² is an electron withdrawing group or an electron donating group,and R⁵ is a chelator group capable of forming a complex with a zinccation.

In particular embodiments, where the R⁵ group of formula 2 is adi-2-picolylamine group, the Zinhbo derivative may be defined by thefollowing formula 3:

where R² is an electron withdrawing group or an electron donating group.

Zinhbo derivatives, when excited with an excitation wavelength, willemit one or more fluorescence responses. In one or more embodiments, theuncomplexed Zinhbo derivative, compared to the complexed Zinhboderivative, has a weak florescence response in the visible region. Inone or more embodiments, the Zinhbo derivative complexed with a zinccation, compared to the uncomplexed Zinhbo derivative, has an increasedflorescence response in the visible region. In one or more embodiments,the Zinhbo derivative complexed with a zinc cation has florescenceresponse in the near infrared region not detectable in the uncomplexedZinhbo derivative. In these or other embodiments, the Zinhbo derivativein the presence of zinc cations, compared to the uncomplexed Zinhboderivative, will produce an increased emission in the visible region,near infrared region, or both the visible and near infrared region.

Zinhbo derivatives when excited with an excitation wavelength will emitone or more fluorescence responses. In one or more embodiments, theuncomplexed Zinhbo derivative will have two florescence response maximain the visible region. In one or more embodiments, the Zinhbo derivativecomplexed with a zinc cation has a single florescence response maxima inthe visible region. In one or more embodiments, the Zinhbo derivativecomplexed with a zinc cation has a florescence response maxima in thenear infrared region. In these or other embodiments, the Zinhboderivative in the presence of zinc cations will produce an emission inthe visible region, near infrared region, or both the visible and nearinfrared region.

A Zinhbo derivative may be prepared through the process 110 of FIG. 2where the reactants 112 are reacted in a alcohol solvent under reflux atstep 114. The alcohol solvent is then evaporated and the product isreacted with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) inmethylene chloride, then purifide, and treated with pyridiumchlorochromate in methylene chloride to yield intermediate product 116.The intermediate product 116 is purified and then reacted with reactant118 in toluene under reflux to make intermediate product 122.Intermediate product 122 is then purified and reacted at step 124 withN-bromosuccinimide to brominate the pendant methyl group. The brominatedpendant methyl group is then reacted with (a) an amine compound thatincludes a group capable of chelating and (b) Na₂CO₃ in dry THF tocreate the R⁵ chelator group capable of forming a complex with a zinccation of intermediate compound 126. The protecting groups are thenremoved to produce at step 128 to produce the Zinbo derivative 130.

In process 110 of FIG. 2, R¹, R², R³, and R⁴ are each individuallyselected from the group consisting of a hydrogen atom, an electronwithdrawing group, and an electron donating group; R⁵ is a chelatorgroup capable of forming a complex with a zinc cation; and Pr is aprotecting group. A protecting group is a group used to protect asensitive group from reacting (in this instance an alcohol group) duringa reaction step in a synthesis of a molecule. The protecting group islater removed. Examples of protecting groups include, but are notlimited to, alkyl groups and silyl groups.

In one or more embodiments, the Zinhbo derivatives may be used to detectthe presence of zinc cations in a test sample. The test sample is asubstance that may contain zinc ions. The Zinhbo derivative is contactedwith the test sample. An excitation wavelength is then applied to thetest sample. In the presence of zinc cations a Zinhbo derivative willform a complex with a zinc ion and the Zinhbo derivative will give anemission wavelength or florescence response that corresponds tocomplexed Zinhbo derivative, thus indicating the presence of zinccations in the test sample. In the absence of zinc cations, the Zinhboderivative will not form a complex and the Zinhbo derivative will givean emission wavelength corresponding to uncomplexed Zinhbo derivatives.

The test sample may be obtained directly from a source to be tested forthe presence of zinc cations, or it may be created by dissolving ordiluting a source to be tested for the presence of zinc cations.

In one or more embodiments, solvents suitable for the creation of testsamples include solvents selected from the group consisting of aqueoussolvents, protic solvents, and organic solvents. Examples of aqueoussolvents include, but are not limited to, water and pH bufferedsolutions. Examples of protic solvents include, but are not limited to,lower alcohols, such as methanol and ethanol. Examples of organicsolvents include, but are not limited to, tetrahydrofuran and methylenechloride.

As a unique feature, the Zinhbo derivative give two emission signalsupon binding to zinc cations; one emission in the visible region, andthe other emission signal in the near infrared region. One advantage ofthis method is that it can be used to test a large number of testsamples all at the same time. This is shown in FIG. 1, showing a method10, wherein a light source 12 directs light 30 (a stimulated emission ofphotons) through a first container 14 holding a test sample 16, a secondcontainer 18 holding a test sample 20 and a third container 22 holding atest sample 24. As seen, the first container 14 and third container 28hold test samples 16 that include zinc, because the light 30 causesfluorescence whose color can be seen visually as at 26 (container 14)and 28 (container 22). The second container 18 does not showfluorescence and thus, the test sample 20 placed therein does notinclude zinc. Although the response of the colored fluorescence signalcan be detected visually by the naked eye, the signals in the nearinfrared region can give more sensitive detection.

In one or more embodiments, the Zinhbo derivative may be used to detectthe presence of zinc cations in vivo. The presence of zinc cations invivo by testing an entire living organism or a portion of an organismthat contains living cells for the presence of zinc cations.

The Zinhbo derivative may be added in vivo, by contacting living cellswith a Zinhbo derivative. In one or more embodiments, the living cellsthat are contacted by the Zinhbo derivative are tissues or part of aliving organism. Examples of methods of contacting living cells with aZinhbo derivative include, but are not limited to, incubating culturedcells with the Zinhbo derivative in a culture medium, injecting Zinhboderivative into living cells. Examples of methods of contacting a livingorganism with a Zinhbo derivative include, but are not limited to,treating a portion of water containing an aquatic animal with a Zinhboderivative. Other methods of contacting a living organism with a Zinhboderivative include, but are not limited, having the living organismingest the Zinhbo derivative and injecting the Zinhbo derivative intothe living organism.

In one or more embodiment, the presence of zinc cations in vivo may befound by exciting the living cells or living organism with an excitationwavelength. If zinc ions are present in vivo the Zinhbo derivative willform a complex with a zinc ion and the Zinhbo derivative will give anemission wavelength, or florescence response, corresponding to complexedZinhbo derivatives and the presence of zinc cations will be detected. Inthe absence of zinc cations, the Zinhbo derivative will not form acomplex and the Zinhbo derivative will give an emission wavelengthcorresponding to uncomplexed Zinhbo derivatives.

In one or more embodiments, the Zinhbo derivative may be used to detectthe presence of free zinc cations in vivo. Free zinc cations refer tothe unbound zinc cations in a cell. Zinc in a cell may be bound toproteins. Zinc imbalance in certain tissues is found to be associatedwith several chronic diseases such as diabetes and Alzheimer's disease.Examples of tissues where free zinc cations may be found includes, butis not limited to, brain, insulin, intestine, and retina.

In particular embodiments, when the Zinhbo derivative is used to detectthe presence of zinc cations or free zinc cations in vivo, it isbeneficial to detect the response emission in the near infraredwavelengths. Detection in the near infrared wavelengths is advantageousbecause wavelengths in the near infrared region can penetrate deeperinto biological tissues. In some embodiments, wavelengths in theinfrared region can penetrate up to about 4 cm of biological tissue. Insome embodiments, wavelengths in the infrared region can penetrate up toabout 3 cm of biological tissue. In one or more embodiments, the Zinhboderivative may be used to detect zinc cations in an organism by scanningan organism with a near infrared light.

The ability of wavelengths in the infrared region to penetratebiological tissue allows for Zinhbo derivatives to be used to scan anorganism for zinc or free zinc cations. In one or more embodiments, anentire organism can be scanned for the presence of zinc cations bydelivering a Zinhbo derivative internally to an organism, exciting theentire organism with an excitation wavelength, and detecting theflorescence response emission. In one or more embodiments, a portionorganism can be scanned for the presence of zinc cations by delivering aZinhbo derivative internally to an organism, exciting a portion of theorganism with an excitation wavelength, and detecting the florescenceresponse emission. By scanning an organism or a portion of an organismfor zinc cations it allows a map of zinc content within the organism tobe determined which may benefit in the treating and research of diseasesassociated with the imbalance of zinc with in the organism's tissues,such as cancer, diabetes and Alzheimer's disease.

The amount of Zinhbo derivative used to detect zinc ions can also bedefined in terms of nM. In one or more embodiments, the amount of Zinhboderivative in solution is 0.01 nM to 100 nM. In other embodiments, theamount of Zinhbo derivative in solution is 0.1 nM to 50 nM. In stillother embodiments, the amount of Zinhbo derivative in solution is 0.2 nMto 5 nM.

In one or more embodiments, the Zinhbo derivatives are excited with anexcitation wavelength from about 400 nm to about 550 nm. In one or moreembodiments, the Zinhbo derivatives are excited with an excitationwavelength from about 430 nm to about 500 nm. In particular embodiment,the Zinhbo derivatives are excited with an excitation wavelength atabout 480 nm.

In one or more embodiments, the Zinhbo derivative complexed with a zinccation has a florescence response emission in the near infrared region.In one or more embodiments, the Zinhbo derivative has a florescenceresponse emission in the range of about 700 to about 900 nm. In one ormore embodiments, the Zinhbo derivative has a florescence responseemission in the range of about 710 to about 800 nm. In these or otherembodiments the Zinhbo derivative has a florescence response emission atabout 760 nm.

In one or more embodiments, the Zinhbo derivative complexed with a zinccation has an increased florescence response emission in the visibleregion. In one or more embodiments, the Zinhbo derivative has aflorescence response emission in the range of about 500 nm to about 700nm. In one or more embodiments, the Zinhbo derivative has a florescenceresponse emission in the range of about 530 nm to about 590 nm. In theseor other embodiments the Zinhbo derivative has a florescence responseemission at about 550 nm.

In one or more embodiments, the Zinhbo derivative will exhibit afluorescence response emission when excited in the visible region. Inthese and other embodiments, the Zinhbo derivative complexed with a zinccation have two or more florescence response emissions in the range ofabout 500 nm to about 900 nm. In these embodiments, the presence of zinccations can be detected by emissions in the visible region, nearinfrared region, or both the visible and near infrared region.

In one or more embodiments, the uncomplexed Zinhbo derivative has aflorescence response emission when excited in the range of about 510 toabout 570 nm. In one or more embodiments, the uncomplexed Zinhboderivative has two florescence response emission maxima in the range ofabout 510 to about 570 nm. In these or other embodiments the Zinhboderivative has a florescence response emission at about 520 nm and aflorescence response emission at about 560 nm.

EXAMPLES

In order to facilitate the in vivo study of zinc-related biology, it isessential to develop a zinc-selective sensor that exhibits bothnear-infrared (NIR) emission and larger Stokes shift. A fluorescentsensor, Zinhbo-5, has been constructed by using bis(benzoxazole) ligandwith 2,2′-dipicolylamine (DPA) as metal ion receptor. In aqueoussolution, Zinhbo-5 exhibits high sensitivity (Kd=2.58 nM²) andselectivity for Zn²⁺ cation, revealing about 14-fold fluorescenceenhancement upon zinc binding to give green emission. Remarkably, Zn²⁺binding to Zinhbo-5 switches on the excited state intramolecular protontransfer (ESIPT), producing the desirable near-infrared region (over 710nm) with large Stokes shift (ca. 240 nm). The new probe is demonstratedto be useful for in vivo imaging of the intracellular Zn²⁺ ion. TheZinhbo-5 is also useful for detecting zinc ion distribution during thedevelopment of living zebrafish embryos.

With reference to FIG. 5, our recent study illustrates that2,5-bis(benzoxazol-2′-yl)benzene-1,4-diol derivative (Zinhbo-1) can giveturn-on ESIPT fluorescence upon addition of zinc(II) cation. A uniquefeature in Zinhbo-1 is that the sensor molecule contains two HBO units.In its zinc complex 4, only one HBO unit is used to bind Zn²⁺ cation,while the other HBO is retained for ESIPT. Zinc binding not only turnson the fluorescence but also enables the ESIPT emission with a largeStokes shift (ca 230 nm). The NIR emission band from Zinhbo-1-Zn²⁺complex (at ˜710 nm), however, is very weak, preventing its practicalapplications. In the Zn²⁺ complex 4 (FIG. 3), the electron flow for theESIPT on the remaining HBO unit is indicated by arrows. The alkyl groupat “position 4” of the benzoxazole unit appears to exert oppositeelectronic effect on the benzoxazole “nitrogen” atom (also indicated byappropriate arrows). This adverse effect could be partially reduced bymoving the alkyl to “position 5” as shown in the compound 5. In order totest this hypothesis, we designed the synthesis of Zinhbo-5. Uponaddition of Zn²⁺ cation, Zinhbo-5 exhibits remarkably strong fluorescentturn-on of the desirable NIR signal along with large Stokes' shift,thereby enabling this dye's practical applications in biological cells.

Results and Discussion

Sensor Zinhbo-5 was synthesized as shown in FIG. 6, which includes asequential construction of two HBO units as shown in compound 7 and 8.With reference to FIG. 6, the reagents and reaction conditions were asfollows: a) (i) MeOH, 5 A Molecular Sieves, Reflux, 81.8%; (ii) DDQ,CH₂Cl₂, 86.9%; b) PCC, CH₂Cl₂; c) (i) Toluene, Reflux, (ii) iodobenzenediacetate (IBD), CH₂Cl₂, 64.6%; d) NBS, AIBN, CCl₄; e) DPA, Na₂CO₃, THF,12.1%; f) BBr₃, CH₂Cl₂, 17.0%. The sensor includes the di-2-picolylamine(DPA) group near a HBO, since DPA is a classical membrane-permeablechelator with high selectivity for Zn²⁺ over alkali and alkaline-earthmetal ions (occurring in higher concentrations) in biological samples.

Zinc Binding of Zinhbo-5 Ligand-to-Metal Ratio.

In ethanol, Zinhbo-5 exhibited an absorption band at (λ_(max)≈412 nm).Upon addition of Zn²⁺, the absorption band was progressively decreased,which was accompanied with a new band at about 465 nm (FIG. 7). Thelarge spectral bathochromic shift indicated the deprotonation, as aconsequence of Zn²⁺-binding to phenol. A clear isobestic point at about427 nm suggested that Zn²⁺-binding produced only one new chemicalspecies. High resolution mass spectroscopy (HRMS) detected the mass1409.238, corresponding to [2(Zinhbo-5)+ClO₄+Na+Zn]+(the calcd mass forC₇₄H₆₅ClN₁₀O₁₂Zn: 1409.366). On the basis of Job plot and HRMS data, thereaction of Zinhbo-5 and Zn²⁺ was assumed to give complex 9 with aligand-to-metal ratio of 2:1 (FIG. 9). The ligand-to-metal ratio of 2:1is also supported by titration experiment in ¹H NMR (FIG. 8).

A mixture of Zinhbo-5 and Zn(ClO₄)₂ (1:1 molar ratio) in ethanol wasrefluxed for 10 min. After slow evaporation of solvent at roomtemperature, the product gave needle-like crystals. Crystal structurerevealed the ligand-to-metal ratio of 1:1 (FIG. 10). Although a HBOderivative could have two possible rotamers that are interconvertible,the crystal 10 revealed only one, in which the free hydroxy group (labedas O(2) atom) was on the same side as the benzoxazole nitrogen N(1),thereby facilitating the desirable ESIPT. The structure showed that theZn2+ cation was penta-coordinated. The crystal 10 gave a rather simple¹H NMR spectrum, revealing two methylene protons as singlets at 4.24 and4.20 ppm (1:2 ratio) (FIG. 11), which is consistent with the reportedZinhbo-1-Zn²⁺ complex.

The reaction of Zinhbo-5 and Zn²⁺ (1:1 molar ratio) was further examinedby ¹H NMR at room temperature (FIG. 11). After addition of Zn²⁺, theresonance signal at ˜8.75 ppm (Ha) gradually disappeared as aconsequence of zinc complexation. A quartet signal centered at ˜4.24 ppmwas clearly observed after mixing for 1 day, indicating the formation ofcomplex 9. Part of the quartet signal could be seen when Zinhbo-5 andZn²⁺ were mixed for 3 minutes. After mixing for 3 days, no trace ofcomplex 10 was observed, on the basis of chemical shifts for the singletaromatic proton at 8.94 ppm (versus 8.89 ppm), and singlet methyleneprotons at 4.24 and 4.20 ppm. Therefore, mixing Zinhbo-5 with Zn²⁺gradually formed complex 9 as the kinetically favored product at roomtemperature, whose conversion to the thermodynamically more stable 10can only occur at a higher temperature.

Fluorescence of Zinhbo-5-Zn²⁺

Free ligand Zinhbo-5 gave only weak green fluorescence at 520 and 560nm. The green fluorescence signal was increased gradually upon additionof Zn²⁺ (FIGS. 12 and 13). In addition, a new emission peak at ˜763 nmoccurred, which has a very large Stokes shift (˜285 nm). The newemission band was attributed to the keto form arising from the ESIPTprocess (FIGS. 14 and 15). Comparison between Zinhbo-5 and Zinhbo-1showed that the former gave quite stronger NIR emission (at ˜763 nm)upon zinc binding (FIG. 14). The stronger NIR emission (at ˜763 nm) fromZinhbo-5 indicated that the keto emission, enabled through zinc binding,is quite sensitive to the substitution position. This is because thesubstituent at the 5-position of benzoxazole unit exerts less electronicinterferences to the anticipated ESIPT process (as shown in structure5). Interestingly, the desirable NIR emission was also affected by thecomplex structure. The NIR emission band (at 763 nm) from the simplemixing of Zinhbo-5 with Zn²⁺”, which gave predominant 9, was noticeablystronger than that from the crystal 10 with well defined 1:1 ratio ofZinhbo-5 to Zn²⁺ ion. The stronger ESIPT from 9 could be associated withthe increased rigidity, when two ligands were assembled together viachelation with Zn²⁺ cation.

Excitation spectra of Zinhbo-5+Zn²⁺ in THF was monitored at 526 and 720nm (FIG. 16). The excitation maximum of Zinhbo-5+Zn²⁺ revealed peaks at˜460 nm, corresponding to the absorption λ_(max) of the deprotonatedZinhbo-5 in the complex. The result indicated that the emission signalsat ˜540 and ˜770 nm were originated from the same species, i.e.Zinhbo-5-Zn²⁺ complex, that gave both enol and keto emission.

The keto emission of Zinhbo-5-Zn²⁺ complex was quite responsive tosolvent polarity (FIG. 17). Non-polar solvents such as ether appeared todisfavor the ESIPT. In DMSO (a polar aprotic solvent), Zn²⁺ could besolvated to reduce the metal binding to phenoxide, thereby inhibitingthe ESIPT. Interestingly, the ESIPT signals were stronger in the proticsolvents (EtOH and MeOH). A possible rational is that protic solventsinteracted with phenoxide by hydrogen bonding, thereby further reducingthe negative charge impact of the phenoxide oxygen and enhancing theESIPT.

In HEPES buffer solution with 50% EtOH, the weak fluorescence of freeligand Zinhbo-5 (ψ_(fl)=0.081) was increased notably to Φ_(fl)=0.17,upon addition of one equivalent Zn2+ cation. The NIR emission intensityat ˜710 nm was linearly proportional to the Zn²⁺ concentration (linearlydependent coefficient: R²=0.9982) (FIGS. 18 and 19). The resultindicated that sensor Zinhbo-5 could be potentially useful forquantitative detection of Zn²⁺ concentration. The association constantsK₁₁ and K₁₂ were determined by a nonlinear least-squares analysis offluorescence intensity versus Zn²⁺ ion concentration to be 3.21×10⁵ M⁻¹and 3.88×10¹⁰ M⁻², respectively.

Potential Use in Zinc Imaging

Detection of Intracellular Zn²⁺ with Zinhbo-5

The Zinhbo-5 sensor was applied on Hela cells, in order to examinewhether the probe could be useful in living systems under a confocallaser scanning microscopy. Cultured Hela cells were incubated withZinhbo-5 in culture medium for 30 min at 37° C., and very weakfluorescence of Zinhbo-5 inside the living Hela cells was observed.After three times washing with PBS buffer, the cells were incubated withZnCl₂ (10 μM) in the medium for another 30 min at 37° C., and thefluorescence in living cells was much brighter (FIG. 20, at D). Abright-field transmission image of cells treated with Zinhbo-5 and Zn²⁺confirmed that the cells were viable throughout the imaging experiments(FIG. 20, at C). Subsequent addition of the high-affinity zinc chelatorN,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) reverted theemission intensity to that of the zinc-free system (FIG. 20, at F).Zinhbo-5 can also be applied to MCF-7 breast cancer cell, giving thesimilar results. The results demonstrated that Zinhbo-5 wascell-permeable and primarily nontoxic to the cell culture and useful forthe imaging of Zn²⁺ in living cells.

Imaging of Zn²⁺ in Zebrafish

The most attractive application for zinc probes is sensing Zn²⁺ inliving organisms. Our ability to discover the fundamental roles of Zn²⁺is dependent on the tools available to detect the Zn²⁺population/concentration in living biological systems. Zebrafish hasrecently become an important model organism for the study of vertebrateimaging. This is because the embryos of zebrafish are transparent,allowing for clear observation of their organs without the need fordissection. The optical transparancy of zebrafish, therefore, provides aconvenient organism for detection of ions by fluorescence microscopy.Several studies have demonstrated the wide use of zebrafish in detectingZn²⁺ ions. Encouraged by the cell permeability (FIG. 20), Zinhbo-5 wasthus examined in developing zebrafish of various stages by addingZinhbo-5 to fish tank water (treatment lasted for one hour at roomtemperature). At 17 hours post fertilization (hpf), thezinc-concentrated cells appeared as green spots which were distributedthroughout the embryos (FIG. 21, at A and B). At 50 hpf (FIG. 21, atC—F) and 72 hpf (data not shown), the zinc-concentrated cells are mainlylocated in the nose and neuromasts of the lateral line system, similarto expression of MTF-1, a zinc finger protein, in zebrafish embryos. TheZinhbo-5 labeling pattern remained similar in older larvae (82 hpf, FIG.22, at A and D). Zinhbo-5 labeling was almost completely eliminated in82 hpf larvae treated with TPEN, a zinc chelator (FIG. 22, at C, F andJ), confirming that the observed Zinhbo-5 labeling was indeed correlatedto the presence of Zn²⁺ ion. There was no obvious difference in Zinhbo-5labeling in the lateral line structures between larvae treated withexogenous Zn²⁺ (20 μM, FIG. 22, at B, E and I) and untreated larvae(FIG. 22, at A, D and G), suggesting that the fluorescence-spotted arearesulted from endogeneous zinc ions in fish and not from sequestrationof the probe. However, there were more labeling in other parts of thebody in the Zn²⁺ treated larvae (FIG. 22 at I). Although several studieshave demonstrated the use of zebrafish in detecting Zn²⁺ ions, the exactorgan associated with the Zn²⁺ bright dots is not known yet. Herein thein vivo recognition of the zinc-rich organ in zebrafish larva could beseen with higher image quality by using Zinhbo-5. All the resultssuggest that Zinhbo-5 has the potential to become a useful sensor fortracing the Zn²⁺ storage in the specific organs during the developmentof zebrafish. The in vivo results with sensor Zinhbo-5 are somewhatdifferent in light of the previously reported data where zinc wasdetected using fluorescent sensors in zebrafish. In those studies,fluorescent labeling was concentrated in the heart ventricle of embryos(50 hpf), and scattered throughout the body in larvae (4-5-day old).Whereas Zinhbo-5 labeling is confined mainly to the nose and neuromastsof the lateral line system during this period.

FIG. 21: Zinhbo-5 Labeling of Developing Zebrafish at 17 and 50 HoursPost Fertilization (hpf).

All images show lateral views of live embryos (anterior to the left anddorsal up). The labeled cells are scattered throughout the embryo at 17hpf (panels A and B). Panel B is a higher magnification of the mid-trunkregion of the embryo shown in panel A. Arrowheads in panel B indicateseveral labeled cells. Zinhbo-5 labeling becomes more restricted asdevelopment proceeds. At 50 hpf, Most labeled cells are concentrated inthe nose neuromasts of the lateral line system (panels C-F). Arrowheadsand asterisks in panels C and D point to some neuromasts in theposterior lateral line system on this side and the other side of thebody trunk, respectively. Panels E and F are higher magnifications ofthe head region of the labeled embryos. Embryos in panels D and F hadbeen treated with Zn2+ before Zinhbo-5 labeling. (Abbreviations: io2,infraorbital line neuromast 2; ml1, middle line neuromast 1; n, nose; ofand o2, otic lateral line neuromasts 1 and 2; po1, postorbital neuromast1; so2, supraorbital line neuromast 2).

FIG. 22

Zinhbo-5 labeling is greatly reduced in zebrafish larvae (82 hpf)exposed to TPEN, a zinc chelator. All images show lateral views(anterior to the left and dorsal up) of live zebrafish larvae. PanelsA-C are low magnification images showing almost the entire fish; panelsD-F show higher magnification of the head region, while panels G-J showhigher magnification of the mid-trunk region of the fish. Panels A, Dand G are from larvae labeled with Zinhbo-5. Panels B, E and I are fromlarvae that had been treated with zinc before exposed to Zinhbo-5.Panels C, F and J are from larvae that were simultaneously treated withZinhbo-5 and TPEN. Arrowheads point to some labeled neuromasts, whilearrows indicate a few labeled cells that do not belong to the lateralline system. (Abbreviations: io1 and io3, infraorbital line neuromasts 1and 3; so1, supraorbital line neuromast 1; ye, yolk extension. Otherabbreviations are the same as in FIG. 9).

CONCLUSION

We have designed and synthesized Zinhbo-5, in which a tert-butyl groupwas introduced to perturb the optical behaviors of bis(benzoxazole)chromophore. Selective Zn²⁺-binding enables the mechanism for excitedstate intramolecular proton transfer, thereby leading to NIR emission(˜710-760 nm) with a large Stokes shift (240 nm). With the aid ofspectroscopic studies, the stoichiometry between Zinhbo-5 and Zn²⁺ isfound to be 2:1 in the initially formed complex. The complex of2(Zinhbo-5).Zn²⁺, however, can be transformed into thermodynamicallymore stable Zinhbo-5.Zn²⁺ (with 1:1 ligand to metal ratio). At ambienttemperature, Zinhbo-5+Zn²⁺ complex gives two emission bands (about 546 &710 nm) in aqueous solution, allowing detection by using both visibleand NIR microscopy. The probe can easily penetrate through cellmembranes for in vivo applications. The Zinhbo-5 can also be applied todetect zinc ions during the development of living zebrafish embryos. Incomparison with the known zinc sensors for zebrafish, the developedprobe shows selective recognition of neuromast region, suggesting thatthe new probes might be able to recognize the zinc in certain regionsover the others. The develop probe thus could provide a potentiallyuseful tool for monitoring the distribution of Zn²⁺ in biology.

Experimental Section Materials and General Procedure

All the solvents were of analytic grade. The salt used in stocksolutions of metal ions were Zn(OAc)₂.2H₂O, Co(OAc)₂.4H₂O,Ni(OAc)₂.4H₂O, Cu(OAc)₂.H₂O, MgSO₄, Cd(OAc)₂.6H₂O, Mn(OAc)₂.4H₂O, FeCl₂.4H₂O, CaCl₂, Na₂SO₄, Hg(OAc)₂, AgNO₃, Pb(OAc)₂.3H₂O, KNO₃. Water usedwas ultra filter deionized and purchased from fisher. HEPES(2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid) was fromFisher. NMR spectra were collected on a Varian 300 Gemini spectrometer.Mass spectrometric data were obtained on a HP1100LC/MSD massspectrometry. UV-Vis spectra were acquired on a Hewlett-Packard 8453diode-array spectrometer. Fluorescence spectra were obtained on a HORIBAJobin Yvon NanoLog spectrometer. All pH measurements were made with amodel RS232 meter. The fluorescence quantum yield were obtained usingquinine sulfate as the standard (Φ=0.53, 0.1 M H₂SO₄). Cell fluorescenceimaging were obtained on a Nikon Eclipse TE2000-5 fluorescencemicroscope, and analyzed by MetaFluor software (Universal ImagingCorp.).

Determination of Association Constant

An approximate linear fit formula is used to calculate the singleassociation constant K_(ass1) and K_(ass2):

${\frac{1}{n}\frac{F - F_{0}}{F_{1} - F}} = {{\frac{2k_{1}K_{{ass}\; 2}C_{0}}{F_{1} - F_{0}}\left( {F_{1} - F} \right)} + {K_{{ass}\; 1}C_{0}}}$

where n is the number of equivalents of the added metal ions, F₀ is thefluorescence intensity of the free sensor, and F₁ is the fluorescenceintensity in the presence of excess cation, C₀ is the initialconcentration of Zinhbo-5. K_(ass1) and K_(ass2) can be obtained by alinear analysis of

(F ₁ −F)(x)

versus

$\frac{1}{n}\frac{F - F_{0}}{F_{1} - F}{(y).}$

The individual profile of the fluorescence at 710 nm demonstrated a 2:1stoichiometric host-guest complex for Zinhbo-5 and Zn²⁺, with theoverall association constant being calculated as Kass=3.88×10¹⁰ M⁻²(Kass1=3.21×10⁵ M⁻¹, Kass2=1.21×10⁵ M⁻¹, respectively).

Experimental Details for the Cell Imaging Experiments

Hela cells were cultured in DEME (Invitrogen) supplemented with 10% FCS(Invitrogen). One day before imaging, cells were seeded in 24-wellflat-bottomed plates. The next day, the Hela cells were incubated with10 μM sensor Zinhbo-5 for 0.5 h at 37° C. under 5% CO₂ and then washedwith phosphate-buffered saline (PBS) three times before incubating with10 μM ZnCl₂ for another 0.5 h, cells were rinsed with PBS three timesagain, then the fluorescence imaging of intracelluar Zn2+ was observedunder Nikon eclipase TE2000-5 inverted fluorescence microscopy with a32× objective lens. The Hela cells only incubated with 10 μM Zinhbo-5for 0.5 hour at 37° C. under 5% CO₂ was as a control. For all images,the microscope settings, such as brightness, contrast, and exposure timewere held constant to compare the relative intensity of intracellularZn²⁺ fluorescence.

Experimental Procedure for Synthesis of Zinhbo-52-(4-methylbenzoxazolyl)-5-(hydroxylmethyl)-1,4-bis(hexyloxy)benzene

The sensor Zinhbo-5 can be synthesized as shown in Scheme 1, whichrequires a sequential construction of two benzoxazole units. The firstbenzoxazole unit is constructed by reaction of 2-amino-m-cresol with2,5-bis(hexyloxy)-4-(hydroxymethyl)benzaldehyde in three steps (in 71%yield). The second benzoxazole unit is synthesized by reaction of2-amino-4-(tert-butyl)phenol with4-(4-methylbenzoxazolyl)-2,5-bis(hexyloxy)benzaldehyde.

Herein, the detailed synthetic procedure is described to illustrate thefeatures related to the compound preparations, chemical synthesis, andspectroscopic properties. For the first reaction shown in Scheme 1,activated molecular sieves (5 {acute over (Å)}) was added to 100 mL MeOHin 500 mL flask. Into this flask were charged 2-amino-m-cresol (2.39 g,21.7 mmol) and 2,5-bis(hexyloxy)-4-(hydroxymethyebenzaldehyde (2.54 g,21.7 mmol). The resulting mixture solution was heated to refluxovernight. The solution was cooled to room temperature, filtered, andsolvent was removed using a rotary evaporator. Then solution of DDQ(2,3-dichloro-5,6-dicyano-1,4-benzoquinone) (4.85 g, 21.4 mmol) in 700mL CH₂Cl₂ was added to the above residue. After stirring at roomtemperature for 2 h, the reaction mixture was treated with 400 mLsaturated Na₂CO₃ and methyl chloride to remove the DDQH by-product, thenwashed with water and brine, and dried over Na₂SO₄. After evaporation ofthe solvent, the solid residues were collected and purified on a silicagel column by using an eluant (hexane:CH₃COOEt=15:1). Afterrecrystallization from methanol, the product2-(4-methylbenzoxazolyl)-5-(hydroxylmethyl)-1,4-bis(hexyloxy)benzene wasobtained as an off-white solid (7.8 g, 81.8%), which has the followingspectral properties. ¹H NMR (CDCl₃, 300 MHz, 6): 7.58 (s, 1H), 7.39 (d,J=8.4 Hz, 1H), 7.32 (dd, J=8.4 Hz, J=8.1 Hz, 1H), 7.14 (d, J=7.5 Hz,1H), 7.06 (s, 1H), 4.74 (d, J=6.6 Hz, 2H), 4.07 (tt, J=6.3 Hz, 4H), 2.68(s, 3H), 1.83 (m, 4H), 1.58 (m, 4H), 1.34 (m, 8H), 0.89 (m, 6H). MS(EI): [M+H±]+=356.3, [M+Na⁺]⁺=462.3.

A solution of2-(4-methylbenzoxazolyl)-5-(hydroxylmethyl)-1,4-bis(hexyloxy)benzene(1.99 g, 4.53 mmol) in CH₂Cl₂ (10 mL) was added dropwise to a stirredsolution of freshly prepared pyridium chlorochromate (PCC) (1.46 g, 6.80mmol) in CH₂Cl₂ (100 mL) at room temperature. Anhydrous Et₂O (100 mL)was added, and the mixture was stirred for an additional 2 h. Thereaction mixture was then directly transferred onto the top of a shortsilica gel column. The yellow and highly fluorescent product was thenwashed off the column with CH₂Cl₂, giving2,5-bis(hexyloxy)-4-(hydroxymethyl)benzaldehyde in 86.9% (1.72 g). Theproduct (mp. 48-50° C.) had the following spectral properties. ¹H NMR(CDCl₃, 300 MHz, 6): 10.53 (s, 1H, —CHO), 7.78 (s, 1H), 7.48 (s, 1H),7.40 (d, J=8.4 Hz, 1H), 7.26 (dd, J=7.8 Hz, J=7.8 Hz, 1H), 7.17 (d,J=7.2 Hz, 1H), 4.18 (t, J=6.6 Hz, 2H), 4.12 (t, J=6.0 Hz, 2H), 2.68 (s,3H), 1.87 (q, J=6.3 Hz, 4H), 1.54 (m, 4H), 1.35 (m, 8H), 0.90 (m, 6H).IR(KBr): 1684 cm⁻¹, 1609.5 cm⁻¹, 1534.6 cm⁻¹.

2,5-Bis(hexyloxy)-4-(hydroxymethyebenzaldehyde (4.3 g, 9.8 mmol) and2-amino-4-tert-butylphenol (1.79 g, 10 mmol) were heated to reflux intoluene (30 mL) under an argon atmosphere for 15 h. The solvent wasremoved, MeOH/CH₂Cl₂ (1:1) (200 mL) and iodobenzene diacetate (3.22 g.10 mmol) were added subsequently. The resulting mixture was stirred atroom temperature for 1 h. After removing solvent under reduced pressure,the residue was neutralized with aqueous sodium bicarbonate solution.After extraction with CH₂Cl₂ and purification on a silica gel column,the product2-(2,5-bis(hexyloxy)-4-(4-methylbenzo[d]oxazol-2-yl)phenyl)-5-(tert-butyl)benzo[d]oxazolwas obtained (3.7 g, 64.6%). ¹H NMR (CDCl₃, 300 MHz, 6): 7.86 (s, 2H),7.84 (s, 1H), 7.49 (d, J=8.4 Hz, 1H), 7.45 (s, 1H), 7.40 (d, J=7.2 Hz,1H), 7.25 (dd, J=7.8 Hz, J=7.8 Hz, 1H), 7.15 (d, J=7.5 Hz, 1H), 4.21(tt, J=6.3 Hz, 4H), 2.69 (s, 3H), 1.96-1.86 (m, 4H), 1.63-1.55 (m, 4H),1.40 (s, 9H), 1.38-1.33 (m, 8H), 0.93-0.87 (m, 6H). ¹³C NMR (CDCl₃, 75MHz, δ ppm): 169.50, 161.84, 160.56, 152.07, 152.04, 152.02, 150.68,149.13, 148.18, 141.90, 141.44, 139.65, 130.87, 125.19, 123.22, 120.21,120.03, 116.75, 116.54, 116.31, 109.95, 108.01, 94.81, 70.29, 51.84,37.80, 35.14, 31.90, 29.60, 28.77, 26.98, 22.90, 16.77, 14.30. HRMS(ESI+) m/z: (M+H)⁺ calcd for C₃₇H₄₇N₂O₄, 583.3536; found: 583.3540.

A mixture of2-(2,5-bis(hexyloxy)-4-(4-methylbenzo[d]oxazol-2-yl)phenyl)-5-(tert-butyl)benzo[d]oxazol(3.7 g, 6.35 mmol), N-bromosuccinimide (NBS) (1.13 g, 6.35 mmol), andAIBN (0.1 g, 0.6 mmol) in CCl₄ (40 mL) was heated to reflux for 15 hunder dry argon atmosphere. The reaction mixture was cooled to 0° C. andthe precipitate was removed by filtration while maintaining thetemperature at 0° C. After the solvent was evaporated,4-(bromomethyl)-2-(4-(5-(tert-butyl)benzo[d]oxazol-2-yl)-2,5-bis(hexyloxy)phenyl)benzo[d]oxazolewas obtained as an oil, which was used for next step without furtherpurification.

A mixture of4-(bromomethyl)-2-(4-(5-(tert-butyl)benzo[d]oxazol-2-yl)-2,5-bis(hexyloxy)phenyl)benzo[d]oxazole(4.2 g, 6.35 mol), di-2-picolylamine (DPA) (3.78 g, 190 mmol), andNa₂CO₃ (6.73 g, 630 mmol) in dry THF (30 mL) was refluxed overnight. Theresulting mixture solution was cooled to room temperature and thesolvent was removed, water (50 mL) was added to the residues, followedby extraction with CH₂Cl₂. The combined organic phase was washed withbrine, dried over MgSO₄, and concentrated on a rotatory evaporator.After purification on a silica gel column, the product was obtained as ayellow oil (0.6 g, 12.1%). ¹H NMR (CDCl₃, 300 MHz, 6): 8.36 (d, J=4.8Hz, 2H), 7.76 (d, J=6.3 Hz, 2H), 7.72 (s, 2H), 7.61 (d, J=8.1 Hz, 2H),7.48 (dd, J=6.9 Hz, J=7.2 Hz, 2H), 7.36-7.22 (m, 2H), 6.98-6.97 (m, 4H),4.13 (s, 2H), 4.06 (m, 4H), 3.83 (s, 4H), 1.96-1.86 (m, 4H), 1.75 (m,4H), 1.46 (m, 4H), 1.25 (s, 9H), 1.21 (m, 8H), 0.75 (m, 6H). ¹³C NMR(CDCl₃, 75 MHz, δ ppm): 177.71, 173.49, 158.38, 150.13, 149.03, 141.32,136.56, 131.19, 122.05, 120.16, 116.66, 109.22, 93.68, 70.14, 67.72,64.58, 60.33, 59.71, 54.22, 53.36, 51.55, 38.50, 35.05, 31.92, 31.70,29.45, 27.70, 25.85, 22.79, 14.26. HRMS (ESI+) m/z: (M+H)⁺ calcd forC₃₄H₂₇N₅O₄, 780.4489; found: 780.4483.

A solution of1-(2-(4-(5-(tert-butyl)benzo[d]oxazol-2-yl)-2,5-bis(hexyloxy)phenyl)benzo[d]oxazol-4-yl)-N,N-bis(pyridin-2-ylmethyl)methanamine (0.6g, 0.77 mmol) in 10 mL of dry CH₂Cl₂ was cooled to −78° C. with dryice/acetone. BBr₃ in CH₂Cl₂ (1 M, 3 mL) was added dropwise under anargon atmosphere. The reaction mixture was stirred at −78° C. for 2 hand then at room temperature overnight. Following the addition of 4.0 mldistilled H₂O, the reaction mixture was stirred for additional 2 h andpoured into a mixture of water (30 mL) and CH₂Cl₂ (30 mL). The organiclayer was separated, and the aqueous layer was extracted twice withCH₂Cl₂ (60 ml). The combined organic layer was washed with brine anddried over anhydrous MgSO₄. After removal of the solvent on a rotaryevaporator, the crude product was purified by recrystallization fromMeOH/acetone (1:1), which give Zinhbo-5 as yellow power solid (0.08 g,17.0%). ¹H NMR (CD₃OD, 300 MHz, 6): 8.76 (d, J=5.7 Hz, 2H), 8.43 (t,J=7.8 Hz, 2H), 8.13 (d, J=8.4 Hz, 2H), 7.89 (t, J=6.3 Hz, 2H), 7.82 (s,1H), 7.75 (d, J=8.7 Hz, 2H), 7.63 (m, 3H), 7.39 (m, 2H), 4.46 (s, 4H),4.27 (s, 2H), 1.42 (s, 9H). ¹H NMR (CDCl₃, 300 MHz, δ ppm): 11.16 (s,1H), 10.34 (s, 1H), 8.86 (d, J=5.4 Hz, 2H), 8.42 (s, 2H), 8.30 (s, 2H),7.84 (s, 2H), 7.80 (s, 1H), 7.78 (s, 1H), 7.58 (m, 3H), 7.53 (s, 1H),7.50 (s, 1H), 7.24 (dd, J=8.4 Hz, J=7.8 Hz, 1H), 4.76 (s, 4H), 4.37 (s,2H), 1.42 (s, 9H). ¹³C NMR (DMSO-d₆, δ ppm): 31.94, 35.31, 57.33,115.49, 115.66, 116.00, 116.41, 124.60, 124.89, 125.43, 125.45, 126.64,140.18, 147.67, 149.17, 149.60, 150.42, 150.44, 153.67, 161.46, 161.62.HRMS (ESI+) m/z: calcd for C₃₇H₃₄N₅O₄, 612.2611; found: 612.2582 (M)±.

A mixture of Zinhbo-5 and Zn(ClO₄)₂ (1:1 molar ratio) in ethanol wasrefluxed for 10 min. After slow evaporation of solvent at roomtemperature, the product gave needle-like crystals. Crystal structurerevealed the ligand-to-metal ratio of 1:1 (FIG. 9). The structure showsthat the zinc(II) cation is penta-coordinated in the complex.

1. A method for detecting the presence of zinc ions in solution, themethod comprising the steps of: contacting a solution with a Zinhboderivative defined by the formula:

where R¹ is an electron withdrawing group or a hydrogen atom; R², R³,and R⁴ are each individually selected from the group consisting of ahydrogen atom, an electron withdrawing group, and an electron donatinggroup; and R⁵ is a chelator group capable of forming a complex with azinc cation; exciting the solution with an excitation wavelength; andmeasuring a florescence response emission in at least one of the visibleor near infrared ranges.
 2. The method of claim 1, where the Zinhboderivative includes an R¹, R³, and R⁴ that are hydrogen atoms and an R²that is and electron donating group.
 3. The method of claim 1, where R⁵is a linear polyamine chelator or a cyclic polyamine chelator.
 4. Themethod of claim 1, where the solution includes a solvent selected fromthe group consisting of aqueous solvents, protic solvents, and organicsolvents.
 5. A method for detecting the presence of zinc ions, themethod comprising the steps of: contacting living cells or a livingorganism with a Zinhbo derivative; exciting the living cells with anexcitation wavelength; and measuring a florescence response emission inat least one of the visible or near infrared range.
 6. The method ofclaim 5, where the Zinhbo derivative defined by the formula:

where R¹, R², R³, and R⁴ are each individually selected from the groupconsisting of a hydrogen atom, an electron withdrawing group, and anelectron donating group; and R⁵ is a chelator group capable of forming acomplex with a zinc cation.
 7. The method of claim 6, where the Zinhboderivative includes an R², R³, and R⁴ that are hydrogen atoms and an R¹that is and electron donating group.
 8. The method of claim 7, where theelectron donating group is an alkyl group.
 9. The method of claim 6,where the Zinhbo derivative includes an R¹, R³, and R⁴ that are hydrogenatoms and an R² that is and electron donating group.
 10. The method ofclaim 9, where the electron donating group is an alkyl group.
 11. Themethod of claim 5, where living cells are contacted with a Zinhboderivative and the living cells are cancer cells.
 12. The method ofclaim 5, where a living organism is contacted with a Zinhbo derivative.13. The method of claim 6, wherein said step of contacting living cellsor a living organism includes delivering the Zihnbo derivativeinternally to an organism.
 14. The method of claim 6, where R⁵ is alinear polyamine chelator or a cyclic polyamine chelator.